PROJECT SUMMARY Retinoid X receptor-? (RXR?), a unique member of the nuclear receptor superfamily, acts as a ligand- dependent transcription factor by recruiting coregulators to positively or negatively regulate target gene transcription. RXR? also acts nongenomically in the cytoplasm to crosstalk with different signaling pathways. However, the mechanisms by which RXR? exerts its nongenomic activities remain obscure. We propose that the nongenomic action of RXR? is regulated by a set of cytoplasmic coregulators that act as counterparts to nuclear coregulators, and that the effect of the nongenomic coregulators can be modulated by ligands via a unique binding mechanism. This innovative hypothesis is built upon our published and preliminary results. We previously reported that RXR? is proteolytically cleaved in cancer cells, resulting in an N-terminally-truncated RXR? (tRXR?) protein that resides in the cytoplasm, where it activates phosphoinositide 3-kinase (PI3K) signaling by interacting with its p85? regulatory subunit. Recently, we discovered that an LxxLL motif utilized by genomic coactivators for binding to the coregulator-binding site (CoRBS) of nuclear receptors is present in p85? and required for binding. Our preliminary results also identified two tumor necrosis factor receptor (TNFR)-associated factors, TRAF2 and TRAF6, as potential nongenomic coregulators of tRXR? that regulate its activation of NF-?B. In our effort to identify and characterize small molecules for inhibiting tRXR? activities, we found via structural analysis that Sulindac-derived analogs bound to previously unrecognized sites on tRXR? to stabilize its tetrameric state, leading to dissociation of p85?. We also discovered a small molecule that binds to CoRBS of tRXR? and prevents its interaction with p85?. In this application, we propose a multidisciplinary approach to address our hypothesis, with 4 Aims: (1). Determine whether p85?, TRAF2 and TRAF6 serve as nongenomic coregulators of tRXR?. We will employ mutagenesis coupled with biological analysis to address the role of CoRBSs in tRXR? and cognate binding motifs in p85?, TRAF2, and TRAF6. (2). Define the structural and functional determinants of RXR?/peptide and protein complexes using complementary crystallographic and functional approaches. (3). Design and identify small molecules that regulate the interaction between tRXR? and its nongenomic coregulators. We will optimize compounds for stabilizing tRXR? tetramerization and for directly inhibiting coregulator binding. (4). Study the role of tRXR? and inhibitors in the development of the inflammatory tumor microenvironment using co-culture assays, and in the growth of tumor in nude and transgenic mice, which will also be used to evaluate inhibitors of nongenomic coregulators. Results obtained from this study should unravel fundamental regulatory mechanisms for the nongenomic action of RXR? (and perhaps other nuclear receptors), and may lead to the identification of new RXR? modulators for therapeutic intervention and mechanistic study.